Systematically quantitative proteomics and metabolite profiles offer insight into fruit ripening behavior in Fragaria × ananassa

Abstract

Profound metabolic and proteomic changes involved in the primary and the secondary metabolism are required for the ripeness of fleshy fruit such as strawberries (Fragaria × ananassa). Here we present the quantitative proteomic profiling in parallel with metabolic and transcriptional profiling at five developmental stages of strawberry fruit ripening, and correlations between changes in representative metabolites and the abundance of related proteins were analyzed. Hierarchical clustering analysis of the quantitative proteomic profiling identified 143 proteins in strawberry fruit across five developmental stages. Meanwhile, both protein abundance and gene expression spanned a wide range of roles, such as the primary and the secondary metabolism, defense system, and response to stress stimuli. The decreased abundance of proteins contributed to the carbohydrate metabolism and the up-regulated expression of secondary biosynthetic proteins was found to be positively correlated with the accumulation of primary and secondary metabolites during strawberry development. Moreover, with the same annotations and high homology, the gene function of key genes involved in primary and secondary metabolism (FaTPI, FaPAL, FaMDH and FaME) was confirmed in Nicotiana via the transient expression assay, which provides further evidence for the role of those genes in metabolism of strawberry fruit. The results of the present study may serve as an important resource for the functional analysis of the proteome and offer new perspectives on regulation of fruit quality.

Fig. 1. Sugar and acid metabolites present in strawberry five developmental stages. Data shown are mean ± standard deviation (n = 3). The unit of the compounds presented in the figure was g kg⁻¹ fresh weigh.

Fig. 2. The amino acids detected in strawberry at five developmental stages. Data shown are mean ± standard deviation (n = 3). The unit of concentrations of compounds presented in the figure was g kg⁻¹ fresh weigh. Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartate; Ser, serine; Gln, glutamine; Glu, glutamic acid; Ile, isoleucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

Fig. 3. Fatty acids and flavonoid compounds present in strawberry at five developmental stages. Data shown are mean ± standard deviation (n = 3). The unit of concentrations of compounds presented in the figure was g kg⁻¹ fresh weigh. Cy3glc, 3-O-β-glucopyranosides of cyaniding; MUFA, monounsaturated fatty acids; Mv3glc, 3-O-β-glucopyranosides of malvidin; Pg3glc, 3-O-β-glucopyranosides of pelargonidin; Pn3glc, 3-O-β-glucopyranosides of peonidin; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TFC, total flavonoids compounds; TPC, total phenolic compounds.

Fig. 4. Venn diagram and hierarchical cluster analysis of identified proteins in strawberry fruit at five developmental stages. (A) Venn diagram; (B) hierarchical cluster analysis. Detailed information were listed in Table S2. The red or blue colors indicates differentially altered abundances compared with strawberry proteins at small green (SG) stage.

Fig. 5. Transient expression assay of NtTPI and FaTPI genes in tobacco plants. The VIGS induced gene silencing was performed with 2mDNA1 and Pbinplus-Y35-1.9A. The overexpression of target genes was driven by pCAMBIA1305 with a CaMV 35S promoter. (A) Relative gene expression in 2mDNA1 empty and 2mDNA1-NtTPI infected tobacco plants; (B) changes of primary metabolites in 2mDNA1 empty and 2mDNA1-NtTPI infected tobacco plants (C) relative gene expression in 1305-GFP empty and 1305-GFP-FaTPI infected tobacco plants; (D) changes of primary metabolites in 1305-GFP empty and 1305-GFP-FaTPI infected tobacco plants.

Fig. 6. Transient expression assay of NtPAL and FaPAL genes in tobacco plants. (A) Relative gene expression in 2mDNA1 empty and 2mDNA1-NtMDH infected tobacco plants; (B) changes of phenylalanine contents in 2mDNA1 empty and 2mDNA1-NtPAL infected tobacco plants (C) relative gene expression in 1305-GFP empty and 1305-GFP-FaPAL infected tobacco plants; (D) changes of phenylalanine contents in 1305-GFP empty and 1305-GFP-FaPAL infected tobacco plants, the others were the same with that in Fig. 5.

Fig. 7. Transient expression assay of NtMDH and FaMDH genes in tobacco plants. (A) Relative gene expression in 2mDNA1 empty and 2mDNA1-NtMDH infected tobacco plants; (B) changes of primary metabolites in 2mDNA1 empty and 2mDNA1-NtMDH infected tobacco plants (C) relative gene expression in 1305-GFP empty and 1305-GFP-FaMDH infected tobacco plants; (D) changes of primary metabolites in 1305-GFP empty and 1305-GFP-FaMDH infected tobacco plants, the others were the same with that in Fig. 5.

Fig. 8. Transient expression assay of NtME and FaME genes in tobacco plants. (A) Relative gene expression in 2mDNA1 empty and 2mDNA1-NtME infected tobacco plants; (B) changes of primary metabolites in 2mDNA1 empty and 2mDNA1-NtME infected tobacco plants (C) relative gene expression in 1305-GFP empty and 1305-GFP-FaME infected tobacco plants; (D) changes of primary metabolites in 1305-GFP empty and 1305-GFP-FaME infected tobacco plants, the others were the same with that in Fig. 5.

Fig. 9. Schematic diagram of proteins involved in the primary and the secondary metabolism in strawberry at different stages. The red and blue lines indicate upregulated and downregulated expression with developmental stage, respectively.

Fig. 10. Schematic diagram of proteins involved in volatiles and flavonoids in strawberry fruit at different stages. (A) Volatiles compounds; (B) flavonoid compounds. The red and blue lines indicate upregulated and downregulated expression with developmental stage, respectively.